ABSTRACT HIV infection has a profound impact on the nervous system, often leading to HIV-associated cognitive disorder (HAND). The viral infection is effectively contained by combination antiretroviral therapy (cART); however, neurocognitive dysfunction remains widespread in HIV- infected individuals even upon viral suppression. The advent of cART has changed the spectrum of HAND, with deficits in learning and memory and executive function becoming more prevalent. An alarming possibility is that cART or its components, while extending the lives of HIV subjects, may contribute to certain features of the modern-day manifestations of HAND. Notably, cART- era behavioral deficits include those that are thought to depend on new neurons, produced in the adult hippocampus from neural stem cells. Strikingly, the possibility that cART itself, or its combination with viral infection, may contribute to HAND by affecting production of new neurons and their connections has not yet been addressed. This application has been designed to assess the feasibility of investigating changes in connections established by newborn neurons as a possible contributor to HAND. Here, we propose to apply new methods, which we have recently developed, to determine the effects of cART, HIV-1 infection, and the cART/HIV combination on the birth of new hippocampal neurons and on the connections they establish with the short- and long-distance partners. This innovative proposal is based on the joint expertise of two groups, one specializing in the studies of adult neurogenesis and the other ? in HIV biology. In our first Specific Aim, we will use a novel reporter Nestin-H2B-GFP mouse line and NSGW-huCD34+, a humanized mouse model of HIV-1 infection, and a new technique for triple S phase cell cycle analysis to determine the effects of cART and its components on key parameters of adult hippocampal neurogenesis and the fates of neural stem cells. We will next use dual-virus (retrovirus and rabies virus) labeling technique to determine the cART-induced changes in monosynaptic partners of new hippocampal neurons. Assisted by the conclusions and optimized protocols from this aim, we will proceed with the studies of HIV infection in the second Specific Aim. Here, we will use NSGW-huCD34+ mice to determine the key changes that HIV-1 infection and HIV/cART combination have on neural stem cells and production of new neurons and on the circuitry established by these neurons. The results of this project will be the first step towards our overarching goal of determining neuronal circuitry compromised by HIV-1 in the context of cART and identifying new targets for mitigating the cognitive consequences of HIV infection.